Language：Japanese   Publisher：北海道外科学会  

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Language：Japanese   Publisher：北海道外科学会  

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Language：English   Publishing type：Research paper (scientific journal)  

Generally, the thickness of tubular tissues formed from silicone rods through encapsulation of the foreign-body reaction is less than approximately 0.2 mm. On the other hand, it is unclear how hollow cylindrical molds can provide thick tubular tissues, known as Biotubes, with a thickness exceeding 1 mm, during in-body tissue architecture (iBTA) using encapsulation. In this study, histological and structural analyses were performed to understand the reason for the formation of thick mold-based Biotubes. Molds were assembled with a gap between a silicone rod and a stainless-steel cylinder and were embedded into the dorsal subcutaneous pouches of beagles for 2 or 4 weeks. Thick Biotubes were obtained from the harvested mold. The histological analysis showed that the lumen side of the thick Biotubes consisted primarily of type I collagen fibers and α-smooth muscle actin-positive cells, similar to the original rod-based thin Biotubes formed only from silicone rods. Interestingly, the outer region of the thick Biotubes was an immature connective tissue consisting of type III collagen, including primitive somatic stem cells expressing CD90 and SSEA4. These stem cells may have contributed to the formation of the thick-walled Biotubes by differentiating into other cell types and through growth factor production. Because of the potential tissue-repair ability of these stem cells, iBTA could be useful for elucidating the regeneration process, remodeling the physiology/pathology of tissue defects/damage, and cell acquisition. This technology can provide autologous stem cells without in vitro cell culture. Moreover, thick-walled Biotubes may be useful as an alternative stem cell-containing material in regenerative medicine.

DOI： 10.1016/j.jbiosc.2021.07.003

PubMed

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Language：Japanese   Publisher：北海道外科学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本人工臓器学会  

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Language：Japanese   Publisher：(一社)日本人工臓器学会  

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Language：English   Publishing type：Research paper (scientific journal)  

Aiming to increase the content of type 2 collagen in scaffold-free cartilage-like cell sheets prepared using human bone marrow mesenchymal stem cells, the effect of several kinds of additives in a chondrogenic medium was investigated. Addition of ascorbic acid 2 phosphate (VCP) at a high concentration (250 µg/ml) and type 1 atelocollagen (5 µg/ml) increased the accumulation of type 2 collagen by fourfold and twofold, respectively. On the other hand, an antioxidant, glutathione showed no such effect. The synergistic effect of VCP and type 1 atelocollagen resulted in an eightfold increase in the accumulation level of type 2 collagen. Furthermore, the gene expression level of type 2 collagen increased and that of matrix metalloproteinase-13 (MMP-13) decreased to approximately one-third of the control. The increase in type 2 collagen accumulation in the scaffold-free cartilage-like cell sheet might be due to not only the enhancement of the synthesis but also the suppression of the degradation of type 2 collagen by MMP-13.

DOI： 10.1007/s10616-015-9924-3

PubMed

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Language：English  

OBJECTIVE: The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. DESIGN: (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. RESULTS: The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. CONCLUSIONS: These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

DOI： 10.1177/1947603515627342

PubMed

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Language：English   Publisher：(公社)日本生物工学会  

異種由来成分を含まずかつ足場材を用いない軟骨様円盤状細胞シートの無収縮調整のためのヒト骨髄由来間葉系幹細胞(MSC)培養系を開発した。MSCは、TGF-β3、IGF-1、デキサメタゾンを含有する無血清軟骨形成分化培地または他の修飾培地を用いて、セルカルチャーインサート(0.3cm2、細孔径0.4μm、細孔密度1.0×10^8/cm2)内で37℃の5%炭酸ガス下で3週間培養した。シート厚、軟骨特異的遺伝子発現、軟骨様細胞外マトリックス(ECM)蓄積を測定した。さらに、細胞シートのセクションをアルシアンブルーで染色した。無血清軟骨形成分化培地と増殖培地(10%ウシ胎仔血清[FCS])から成る新規混合培地は、細胞シートの収縮を防ぎ、円盤状細胞シートの作製が可能であった。細胞シートの深さは約0.7mmで、遺伝子発現レベルは正常なヒト軟骨細胞よりも高かった。FCSの代わりにヒト血清を用いたところ、細胞シート収縮が発生せず、シート中の硫酸化グリコサミノグリカン類および2型コラーゲンの蓄積レベル減少もなかった。セルカルチャーインサート中でヒト血清を含む混合培地を用いた、完全に異種由来成分を含まない材料によるMSCの培養は、1.0mmのシート深さを有し正常軟骨よりも高い遺伝子発現レベルを示した。

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Language：English  

Aiming for the clinical application of cartilage regeneration, the xeno-free cultivation method to obtain a scaffold-free cartilage-like disc-shaped cell sheet using mesenchymal stem cells (MSCs) derived from human bone marrow without the shrinkage of the sheet was investigated. MSCs were inoculated into Cell Culture Insert (0.3 cm(2), pore size; 0.4 μm, pore density; 1.0 × 10(8)/cm(2)) using serum-free chondrogenic differentiation medium containing TGF-β3, IGF-1 and dexamethasone or other modified media, and cultured at 37 °C in 5% CO2 for 3 weeks. Sheet thickness, cartilage specific genes expression, ECM accumulation were determined, and the sections of sheets were stained with alcian blue. A novel mixed medium consisting of a growth medium (10% FCS) with a serum-free chondrogenic differentiation medium could prevent the shrinkage of the sheet and produced a disc-shaped cell sheet. The depth of the sheet was approximately 0.7 mm and the gene expression levels were higher than those in cells in normal human cartilage. The use of human serum instead of FCS did not cause shrinkage and did not decrease the accumulation levels of sGAG and type 2 collagen in the sheet. The cultivation of MSCs grown with completely xeno-free materials using the mixed medium containing human serum in a cell culture insert showed a sheet depth of 1.0 mm and gene expression levels higher than those in normal cartilage. The scaffold-free and xeno-free cartilage-like cell sheet was successfully formed without shrinkage using human bone marrow MSCs and the chondrogenic differentiation medium containing human serum.

DOI： 10.1016/j.jbiosc.2013.05.019

PubMed

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The cell-specific production rate of melanoma inhibitory activity was quantitatively proportional to the aggrecan gene expression level in the early and middle phase during not only the redifferentiation culture of human cartilage chondrocytes but also the differentiation culture of human bone marrow mesenchymal stem cells to chondrocytes.

DOI： 10.1016/j.jbiosc.2010.12.023

PubMed

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Language：English   Publisher：(公社)日本生物工学会  

軟骨細胞が分泌するmelanoma inhibitory activity(MIA)の産生速度を測定し、軟骨細胞のマーカーであるアグレカン遺伝子発現レベルとの関係を調べた。ヒト関節から採取した軟骨細胞のペレットを培養し、1週間ごとに上清を採取して軟骨細胞の再分化の過程のMIA濃度を測定すると共に、ペレット細胞のアグレカン遺伝子発現レベルを測定した。ヒトの間葉系幹細胞(MSC)は腸骨稜から採取し、MSCを培養して軟骨細胞への分化に伴うMIA産生速度とアグレカン遺伝子発現レベルを測定した。その結果、ヒト軟骨細胞の再分化とMSCから軟骨細胞への分化の過程において、それぞれの初期から中期にかけて、細胞特異的MIA産生速度はアグレカン遺伝子発現と定量的に比例することが明らかになった。

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：ELSEVIER  

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Language：Japanese   Publisher：(一社)日本人工臓器学会  

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Authorship：Lead author   Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese   Publisher：(一社)日本人工臓器学会  

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Language：Japanese   Publisher：(NPO)日本小児循環器学会  

J-GLOBAL

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Language：Japanese   Publisher：北海道外科学会  

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Language：Japanese  

CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：MARY ANN LIEBERT, INC  

Web of Science

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Language：Japanese   Publisher：(公社)日本整形外科学会  

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Language：Japanese   Publisher：(公社)日本生物工学会  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生物工学会  

J-GLOBAL

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Language：Japanese   Publisher：(一社)日本関節鏡・膝・スポーツ整形外科学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCI LTD  

Web of Science

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Language：Japanese   Publisher：(公社)日本生物工学会  

J-GLOBAL

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Language：Japanese   Publisher：(公社)日本生物工学会  

J-GLOBAL

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Event date： 2023.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京都  

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Event date： 2023.11

Language：Japanese   Presentation type：Poster presentation  

Venue：沖縄県北谷町  

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Event date： 2023.9

Language：English   Presentation type：Oral presentation (general)  

Venue：Kuala Lumpur, Malaysia  

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Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋  

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Event date： 2023.3

Language：English   Presentation type：Symposium, workshop panel (nominated)  

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Event date： 2023.2

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：愛媛県民文化会館  

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Event date： 2022.11

Language：Japanese   Presentation type：Poster presentation  

Venue：愛媛県民文化会館  

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Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：愛媛県民文化会館  

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Event date： 2022.11

Language：Japanese   Presentation type：Poster presentation  

Venue：愛媛県民文化会館  

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Event date： 2022.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：愛媛県民文化会館  

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Event date： 2022.4

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2022.2

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2022.2

Language：Japanese   Presentation type：Oral presentation (general)  

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Event date： 2022.2

Language：Japanese   Presentation type：Oral presentation (general)  

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Applicant：バイオチューブ株式会社

Application no：特願2019-128790  Date applied：2019.7

Announcement no：特開2021-013480  Date announced：2021.2

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Applicant：公益財団法人先端医療振興財団

Application no：特願2016-101201  Date applied：2016.5

Announcement no：特開2017-206476  Date announced：2017.11

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Application no：2016-101201  Date applied：2016.5

Announcement no：2017-206476  Date announced：2017.11

Country of applicant：Domestic  

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座長

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実行委員（表彰担当）

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座長

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